One of the major limiting steps in the determination of three-dimensional structures of macromolecules by the technique of X-ray diffraction is the ability to grow relatively large (0.5 mm) high-quality (diffraction limit of 3 angstrom units or better) crystals of the macromolecules of interest. A knowledge of the three-dimensional structures of various enzymes has great potential in the area of drug design, i.e., development of new enzyme inhibitors, and is of fundamental importance in the field of molecular biology.
Recently, researchers involved in protein crystal growth have reported growing protein crystals in a microgravity environment that are many times larger than similar crystals grown in a gravity environment. They used a method known as diffusive mixing, wherein a solution containing dissolved protein is allowed to mix with a solution containing a dissolved solvent and precipitating agent. As there are no disruptive convection currents in a microgravity environment, mixing of the two solutions is achieved by diffusion, allowing a slower approach to the critical supersaturation of the dissolved protein, causing larger crystals to be grown. For more information on growing protein crystals in a microgravity environment, see "Protein Single Crystal Growth Under Microgravity," by W. Littke and C. John in Science, 1984, Vol, 225, page 203.
Another similar diffusive method of growing crystals is the liquid diffusion method in which a solution containing dissolved protein to be crystallized is placed in a dyalysis bag. This bag is constructed of a semi-permeable membrane which will allow smaller solvent and precipitating agent molecules to pass through but blocks larger protein molecules. This bag is immersed in a solution containing a precipitating agent for the protein, which then diffuses through the bag and lowers the solubility of the protein. As the saturation point of the dissolved protein is approached, crystal nucleation occurs, creating tiny nucleii of crystals around which additional crystalline material is deposited.
Problems with this method are that no provisions are made to control the rate at which critical supersaturation is approached. Thus, the rate of crystal growth cannot be precisely controlled, often resulting in numerous smaller crystals being formed having a structure that is something less than ordered. These crystals do not provide resolution necessary under X-ray crystallography to adequately determine their molecular structure.
One of the most popular crystal growth methods, known as the hanging drop method, involves the placement of a small droplet of protein solution on a glass cover slip, with this cover slip being inverted over a well of solution and sealed. The solution in the well contains a precipitating agent that is also present at a lower concentration in the protein droplet (usually 50% of the reservoir concentration). The function of the precipitating solution is twofold. First, the solution in the well is initially at a lower vapor pressure than the protein droplet. Evaporation then proceeds at a rate fixed by the difference in vapor pressure between the protein droplet and the reservoir and the distance the vapor must diffuse. Second, the precipitating agent lowers the solubility of protein in solution, presumably by competing with the protein for available solvent (usually water).
Thus, as evaporation from the protein droplet progresses, the solution becomes supersaturated with protein. under appropriate conditions of pH, temperature, etc., crystallization of the protein or macromolecule then occurs. The exact conditions for crystallization are established by trial and error, with serendipity often being an important factor. A review and a more in-depth description of the vapor diffusion method, as well as other protein crystallization techniques, may be seen in Preparation and Analysis of Protein Crystals, by A. McPherson (1982), Wiley, New York. Again, problems with this method are a lack of control over the rate at which drop size is changed and the inability to reverse the process to increase drop size.
Applicants' device as described herein controls vapor pressure, in turn determining crystal growth rate, in a crystal growth apparatus utilizing the hanging drop method. It has been shown that nucleation rate and crystal size are highly dependent upon the rate at which critical supersaturation is approached. Slow approach to critical supersaturation shows a marked decrease in crystal nucleation rate and a corresponding increase in crystal size and quality.
It is, therefore, an object of this invention to provide a crystal growth apparatus for allowing researchers to controllably approach critical supersaturation in a hanging drop containing dissolved protein material. This is done by either adding or removing solvent in a vapor phase to or from the hanging drop.